Beneficial Effects of a Protein Rich Diet on Coping Neurotrans-mitter Levels During Ampicillin-Induced Neurotoxicity Compared to Propionic-Acid Induced Autistic Biochemical Features.

This study examined the effects of a protein rich diet on coping neurotransmitter levels in orally administered ampicillin-induced neurotoxic rats compared with propionic acid (PA) models of autism. 40 young male western albino rats were divided into four groups. The first group served as control and received phosphate buffered saline orally; the second group serving as autistic model was treated with oral dose of PA (250 mg/kg body weight/day for 3 days); the third group was treated with the neurotoxic dose of ampicillin (50 mg/kg for three weeks); the fourth group received the same dose of ampicillin and was fed with special protein rich diets. Noradrenaline, dopamine, serotonin glutamate, glutamine and interleukin 6 (IL-6) were measured in the brain homogenate of all tested groups. Specified doses of PA and ampicillin significantly (P<0.001) decreased noradrenaline, dopamine, and serotonin levels when compared to control. Also glutamate, IL-6 levels were significantly (P<0.001) increased in PA treated group while non-significant increase was found in ampicillin treated group. Non-significant increase of glutamine was found in PA treated group with a significant increase in ampicillin treated group. The effects of ampicillin on these parameters were found to be potentiated when the rats were fed on a protein rich diet. Our results end with the conclusion that dietary protein level may be a useful tool to find out a path to restrict neurotransmitter alterations in neurodevelopmental disorders like autism.

has recently been reported to induce persistent biochemical and behavioral autistic features in rat pups (7)(8)(9). Ampicillin (Amp) is believed to exert an inhibitory effect on gamma-aminobutyric acid (GABA) transmission due to its beta-lactam ring structure, which is somewhat similar to the GABA structure (10). Imbalance in GABAergic/ glutamatergic, serotonergic, dopaminergic neurotransmission together with neuroinflammation were recently recorded as the most important signals that are impaired and related to clinical presentation and severity of autism (11). Supplementation with high-protein diets is often used to improve physical status causing an effective reduction in body weight, fat deposition and improving plasma lipid profile (12). Some studies have shown the beneficial effects of high-protein diets on rodent brain such as protecting against cerebral ischemia and reducing apoptosis in the ischemic cortex (13,14). However, little is known regarding the effects of high-protein diet and autism in the presence of antibiotics. The development of animal models of autism is one approach that could help identify the mechanism by which autism develops in humans. Thus, rodent model with autistic features was developed through orally administered neurotoxic dose of PA (15) and Amp (16) and effect of high protein diet in shrinking the neurotoxic effect was analyzed by measuring neurotransmitters.

Experimental animals
The experimental assays for this study were performed on 40 young (approximately 21 days old) male western albino rats (45 to 60 g). Rats were obtained from animal house at the pharmacy college in King Saud University and allowed to drink water ab libitum for a period of one week before stating the treatment.

Experimental design
Animals were randomly assigned to four groups of ten rats each. The first group of rats (n=10) received only phosphate buffered saline and were used as a control group. The second group was given oral neurotoxic doses of PA (250 mg/kg body weight/day for three days) (17) and were referred to as the oral buffered PA-treated group.
The third group received an orogastric dose of ampicillin (50 mg/ kg for three weeks) with standard diet and referred to as the ampicillin group (16). Animals of the last group were given orogastric doses of ampicillin (50 mg/kg for three weeks), and were fed with a high-protein diet for 10 weeks. The Ethics Committee at King Saud University approved the protocol of the present test, in addition, all experiments were performed in accordance with the guidelines of the National Animal Care and Use Committee.

Diets
The control protein diet and the protein enriched diets (corresponding to the amount of casein present) were prepared according to the protocol of the Institutional Animal care and Use committee (18). The diets composition has been shown in Table 1.

Tissue preparation
At the end of the feeding trial, the rats were anesthetized with carbon dioxide and decapitated. The brain was removed from the skull and was

Glutamine and glutamate analysis
Rat brain glutamine (Gln) or glutamate (Glut) were measured independently using ELISA kit, a product of Cusabio. Antibody specific for Gln, or Glut has been pre-coated onto a microplate.
Standards and samples were pipetted into the wells where the respective immobilized antibody could bind any Gln or Glut present. After removing any unbound substances, a biotin-conjugated antibody specific for Gln or Glut was added to the wells.
After washing, avidin conjugated horseradish peroxidase (HRP) was added to the wells.

Statistical analysis
The data were analyzed using the Statistical Pearson's correlations were performed between the measured parameters.    Table 4. Table 5 demonstrates the multiple regression analysis using noradrenaline, dopamine, and     serotonin, and IL6, Gln and Glut as dependent variables.

Discussion
In the present study, a significant decrease in noradrenaline, dopamine, serotonin content with increased levels of Glut and Gln was shown in the brain of the PA model of autism. Our study evaluated the role of protein diet in preventing the neurochemical alterations in brain caused by exposure to Amp ( depends largely on the brain concentra-tions of Ltryptophan, its precursor amino acid. Also, the synthesis of catecholamines (e.g., dopa-mine, norepinephrine) in the brain varies with the availability of the precursor amino acid L-tyrosine.
Protein diet induced changes in blood amino acid concentrations, and as a result, will influence the synthesis of neurotransmitters in the brain.
IL-6 is normally expressed at relatively low levels in the brain (22,23). However, elevated cytokine response is associated with autism and IL-6 has been repeatedly found to be increased in the autistic brain (24,25). However, Amp treatment did not seem to have a major impact on IL6 levels in brain tissue. These results can be supported by the report that Amp is able to decrease the blood level of IL-6 by inhibiting prostaglandin E2 synthesis (27).
Furthermore, it was found that Amp treatment with protein rich diet was able to shrink the IL-6 levels auxiliary; which can be supported by the recent finding that proteins rich diets can reduce the IL-6 levels in blood (28). The significant increase of brain Glut in PA and Amp treated groups can easily be related to ASD features as Glut excitotoxicity is one of the most important mechanisms involved in the etiology of autism.
In addition to the AUC, the specificity and sensitivity values listed in figure 2 and Table 4 demonstrate the possibility of using noradrenaline, dopamine, serotonin and IL-6 as markers of PA and Amp neurotoxicity. All measured parameters demonstrated satisfactory sensitivity and very high specificity, which confirmed that PA and Amp can excite toxicity and neuroinflammation.